Clk2 and B56β mediate insulin-regulated assembly of the PP2A phosphatase holoenzyme complex on Akt

Akt mediates important cellular decisions involved in growth, survival, and metabolism. The mechanisms by which Akt is phosphorylated and activated in response to growth factors or insulin have been extensively studied, but the molecular regulatory components and dynamics of Akt attenuation are poorly understood. Here we show that a downstream target of insulin-induced Akt activation, Clk2, triggers Akt dephosphorylation through the PP2A phosphatase complex. Clk2 phosphorylates the PP2A regulatory subunit B56β (PPP2R5B, B'β), which is a critical regulatory step in the assembly of the PP2A holoenzyme complex on Akt leading to dephosphorylation of both S473 and T308 Akt sites. Since Akt plays a pivotal role in cellular signaling, these results have important implications for our understanding of Akt regulation in many biological processes.     

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.     

Mapping of the regulatory type Iα and catalytic β subunits of cAMP-dependent protein kinase and interleukin 1α and 1β in the pig

The genes coding for the regulatory type I subunit (PRKAR1A) and the catalytic subunit (PRKACB) of cAMP-dependent protein kinase and the genes for interleukin 1 (IL1A) and interleukin 1 (IL1B) were localized in the pig by means of radioactive in situ hybridization. PRKAR1A was mapped to 12p1.4 and PRKARB to 6q3.1 q3.3. The genes for IL1A and IL1B were both assigned to Chromosome (Chr) 3, in the region q1.2 q1.3 and q1.1 q1.4, respectively. The cDNA nucleotide sequences of these porcine genes were compared with those of human, mouse, and cattle. The location of the genes was discussed in relation to the position of their homologous loci in these mammalian species.     

The essential β-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis

Autotransporter biogenesis is dependent upon BamA, a central component of the β-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.     

The association of prostaglandins E2 and F2α with erythropoiesis regulatory factors

Prostaglandins E₂ and F_2α have regulatory roles, respectively, in the potentiation and inhibition of erythropoietin. Antisera to PGF_2α and EIF, an erythropoiesis inhibitory factor, can be used to resolve the reason for improvement in erythropoiesis following renal dialysis. Concurrent loss of PGE₂ with Ep during treatment with neuraminidase suggests that PGE₂ is bound to Ep by way of sialic acid.     

Understanding the molecular basis of MK2-p38α signaling complex assembly: insights into protein-protein interaction by molecular dynamics and free energy studies

The formation of a p38 MAPK and MAPK-activated protein kinase 2 (MK2) signaling complex is physiologically relevant to cellular responses such as the proinflammatory cytokine production. The interaction between p38α isoform and MK2 is of great importance for this signaling. In this study, molecular dynamics simulation and binding free energy calculation were performed on the MK2-p38α signaling complex to investigate the protein-protein interaction between the two proteins. Dynamic domain motion analyses were performed to analyze the conformational changes between the unbound and bound states of proteins during the interaction. The activation loop, αF-I helices, and loops among α helices in the C-lobe of MK2 are found to be highly flexible and exhibit significant changes upon p38α binding. The results also show that after the binding of p38α, the N- and C-terminal domains of MK2 display an opening and twisting motion centered on the activation loop. The molecular mechanics Poisson-Boltzmann and generalized-Born surface area (MM-PB/GBSA) methods were used to calculate binding free energies between MK2 and p38α. The analysis of the components of binding free energy calculation indicates that the van der Waals interaction and the nonpolar solvation energy provide the driving force for the binding process, while the electrostatic interaction contributes critically to the specificity, rather than to MK2-p38α binding affinity. The contribution of each residue at the interaction interface to the binding affinity of MK2 with p38α was also analyzed by free energy decomposition. Several important residues responsible for the protein-protein interaction were also identified.     

Molecular cloning and differential expression patterns of the regulatory subunit B′ gene of PP2A in goldfish, Carassius auratus

It is well established that the protein serine/threonine phosphatase 2A (PP2A) plays very important roles in many different cellular processes, including cell proliferation and differentiation, gene expres-sion, neurotransmission, apoptosis, and aging. PP2A consists of three heterogenic subunits: the scaffold subunit A, the catalytic subunit C, and the regulatory subunit B. While both the scaffold and the catalytic subunits contain only two forms, at least four families of the regulatory subunits, B, B, B′′, and B′′′ have been identified. These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A. To study the functions of the regulatory subunits of PP2A in lower vertebrates, we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit B′δ of PP2A from gold fish, Carassius auratus using 3′-RACE and 5′-RACE cloning strategies. Our results revealed that the full-length B′δ cDNA contains 2415 bp and encodes a protein of 555 amino acids. The B′δ protein displays a very high level of sequence identity with the B′δ regulatory subunit from other species of vertebrates. Regarding its expression pattern, RT-PCR revealed that the highest level of mRNA was detected in brain, a less level detected in liver, spermary, ovary, kidney and gill, and the lowest level detected in the fin. During different developmental stages of gold fish, the highest level of mRNA expression was detected at the stages of two-cell, mul-tiple-cell, blastula and gastrula, and a decreased level of B′δ ?mRNA was detected in other develop-mental stages. At the protein level, the highest expression level of B′δ protein was found in spermary, ovary, brain and heart, a less amount found in liver and the lowest level detected in kidney, gill and fin. Developmentally, B′δ protein was strongly expressed at the stages of two-cell, multiple-cell, blastula, gastrula, neurula, and optic vesicle, and then decreased at the stages of brain differentiation and eye pigmentation. These results suggest that B′δ appears to play a very important role during gold fish development and also in adult tissue homeostasis.     

Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2–GDPNP–tRNA complex.     

The importance of the last strand at the C-terminus in βB2-crystallin stability and assembly

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on βB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of βB2-crystallin Greek-key motif 4. All of the three mutations promoted βB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected βB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of β-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between β-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the β-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of β-crystallins are achieved by the precisely regulated oligomeric equilibrium.     

A critical role for the protein phosphatase 2A B'α regulatory subunit in dephosphorylation of sphingosine kinase 1

Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localization and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits, which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and diverse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B'α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B'α was found to interact with the c-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. siRNA-mediated knockdown of B'α increased SK1 phosphorylation, activity and membrane localization of endogenous SK1. Furthermore, overexpression of B'α blocked agonist-induced translocation of SK1 to the plasma membrane and abrogated SK1-induced neoplastic transformation of NIH3T3 fibroblasts. Thus, the PP2A-B'α holoenzyme appears to function as an important endogenous regulator of SK1.     

The combined inhibition of the CaMKIIδ and calcineurin signaling cascade attenuates IGF-IIR-induced cardiac hypertrophy

Cardiac hypertrophy is a common phenomenon observed in progressive heart disease associated with heart failure. Insulin-like growth factor receptor II (IGF-IIR) has been much implicated in myocardial hypertrophy. Our previous studies have found that increased activities of signaling mediators, such as calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin induces pathological hypertrophy. Given the critical roles played by CaMKII and calcineurin signaling in the progression of maladaptive hypertrophy, we anticipated that inhibition of CaMKII and calcineurin signaling may attenuate IGF-IIR-induced cardiac hypertrophy. The current study, therefore, investigated the effects of IGF-IIR activation on the CaMKII and calcineurin signaling and whether the combinatorial inhibition of the CaMKIIδ and calcineurin signaling could ameliorate IGF-IIR-induced pathological hypertrophy. In the present study, we induced IGF-IIR through the cardiomyocyte-specific transduction of IGFII^Y27L via adeno-associated virus 2 (AAV2) to evaluate its effects on cardiac hypertrophy. Interestingly, it was observed that the activation of IGF-IIR signaling through IGFII^Y27L induces significant hypertrophy of the myocardium and increased cardiac apoptosis and fibrosis. Moreover, we found that Leu²⁷IGF-II significantly induced calcineurin and CaMKII expression. Furthermore and importantly, the combinatorial treatment with CaMKII and calcineurin inhibitors significantly alleviates IGF-IIR-induced hypertrophic responses. Thus, it could be envisaged that the inhibition of IGF-IIR may serve as a promising candidate for attenuating maladaptive hypertrophy. Both calcineurin and CaMKII could be valuable targets for developing treatment strategies against hypertension-induced cardiomyopathies.     

A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

Introduction

Increased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.

Methods

Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.

Results

TNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.

Conclusions

Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.     

The quest for trait convergence and divergence in community assembly: are null‐models the magic wand?

The relevance of neutral versus niche‐based community assembly rules (i.e. the processes sorting species present in a larger geographical region into local communities) remains to be demonstrated in ecology and biogeography. To attempt to do this, a number of complex null models are increasingly being used that compare observed community functional diversity (FD, i.e. the extent of trait dissimilarity between coexisting species) with randomly simulated FD. However, little is known about the performance of these null models in detecting non‐neutral community assembly rules such as trait convergence and divergence of communities (supposedly revealing habitat selection and limiting similarity, respectively). Here, using both simulated and field communities, I show that assembly rule detection varies systematically with the magnitude of the observed FD, so that these null models do not really succeed in breaking down the observed functional relationships between species. This is a particular concern, making detection of community assembly dependent on: (1) the pool of samples considered, and (2) the capacity of observed FD to correctly discriminate these rules. Null models should be more thoroughly described and validated before being considered as a magic wand to reveal assembly patterns.     

Monoubiquitination promotes calpain cleavage of the protein phosphatase 2A (PP2A) regulatory subunit α4, altering PP2A stability and microtubule-associated protein phosphorylation

Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.     

The B56γ3 Regulatory Subunit of Protein Phosphatase 2A (PP2A) Regulates S Phase-specific Nuclear Accumulation of PP2A and the G1 to S Transition

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a scaffold subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). The regulatory B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Here, we demonstrate that the subcellular localization of the B56γ3 regulatory subunit is regulated in a cell cycle-specific manner. Notably, B56γ3 becomes enriched in the nucleus at the G₁/S border and in S phase. The S phase-specific nuclear enrichment of B56γ3 is accompanied by increases of nuclear A and C subunits and nuclear PP2A activity. Overexpression of B56γ3 promotes nuclear localization of the A and C subunits, whereas silencing both B56γ2 and B56γ3 blocks the S phase-specific increase in the nuclear localization and activity of PP2A. In NIH3T3 cells, B56γ3 overexpression reduces p27 phosphorylation at Thr-187, concomitantly elevates p27 protein levels, delays the G₁ to S transition, and retards cell proliferation. Consistently, knockdown of endogenous B56γ3 expression reduces p27 protein levels and increases cell proliferation in HeLa cells. These findings demonstrate that the dynamic nuclear distribution of the B56γ3 regulatory subunit controls nuclear PP2A activity, which regulates cell cycle controllers, such as p27, to restrain cell cycle progression, and may be responsible for the tumor suppressor function of PP2A.